Advanced Light Microscopy Unit

Barcelona, Spain

Advanced Light Microscopy Unit

Barcelona, Spain

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Kopp K.,University of Konstanz | Buntru A.,University of Konstanz | Pils S.,University of Konstanz | Zimmermann T.,Advanced Light Microscopy Unit | And 3 more authors.
Journal of Biological Chemistry | Year: 2012

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a phagocytic receptor on human granulocytes, which mediates the opsonin-independent recognition and internalization of a restricted set of Gram-negative bacteria such as Neisseria gonorrhoeae. In an unbiased screen using a SH2 domain microarray we identified the SH2 domain of growth factor receptor-bound protein 14 (Grb14) as a novel binding partner of CEACAM3. Biochemical assays and microscopic studies demonstrated that the Grb14 SH2 domain promoted the rapid recruitment of this adaptor protein to the immunoreceptor-based activation motif (ITAM)-like sequence within the cytoplasmic domain of CEACAM3. Furthermore, FRET-FLIM analyses confirmed the direct association of Grb14 and CEACAM3 in intact cells at the sites of bacteria-host cell contact. Knockdown of endogenous Grb14 by RNA interference as well as Grb14 overexpression indicate an inhibitory role for this adapter protein in CEACAM3-mediated phagocytosis. Therefore, Grb14 is the first negative regulator of CEACAM3-initiated bacterial phagocytosis and might help to focus granulocyte responses to the subcellular sites of pathogen-host cell contact. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.


Pengo T.,Center for Genomic Regulation | Pengo T.,Advanced Light Microscopy Unit | Holden S.J.,Institute for Physics of Biological Systems | Manley S.,Institute for Physics of Biological Systems
Bioinformatics | Year: 2015

During the past decade, localization microscopy (LM) has transformed into an accessible, commercially available technique for life sciences. However, data processing can be challenging to the non-specialist and care is still needed to produce meaningful results. PALMsiever has been developed to provide a user-friendly means of visualizing, filtering and analyzing LM data. It includes drift correction, clustering, intelligent line profiles, many rendering algorithms and 3D data visualization. It incorporates the main analysis and data processing modalities used by experts in the field, as well as several new features we developed, and makes them broadly accessible. It can easily be extended via plugins and is provided as free of charge open-source software. © The Author 2014. Published by Oxford University Press. All rights reserved.


Carvalho A.,EMBL CRG Systems Biology Research Unit | Carvalho A.,University Pompeu Fabra | Carvalho A.,Pasqual Maragall Foundation and Barcelonabeta Brain Research Center | Menendez D.B.,EMBL CRG Systems Biology Research Unit | And 11 more authors.
ACS Synthetic Biology | Year: 2014

Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin-Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell-cell communication networks in 3D cell culture. © 2013 American Chemical Society.


Grunberg R.,EMBL CRG Systems Biology Research Unit | Grunberg R.,University of Montréal | Burnier J.V.,EMBL CRG Systems Biology Research Unit | Burnier J.V.,University Pompeu Fabra | And 19 more authors.
Nature Methods | Year: 2013

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.


Seitz A.,European Molecular Biology Laboratory | Seitz A.,Ecole Polytechnique Federale de Lausanne | Terjung S.,European Molecular Biology Laboratory | Zimmermann T.,European Molecular Biology Laboratory | And 2 more authors.
Journal of Biomedical Optics | Year: 2012

Fluorescence resonance energy transfer (FRET) efficiency measurements based on acceptor photobleaching of yellow fluorescent protein (YFP) are affected by the fact that bleaching of YFP produces a fluorescent species that is detectable in cyan fluorescent protein (CFP) image channels. The presented quantitative measurement of this conversion makes it possible to correct the obtained FRET signal to increase the accuracy of intensity based CFP/YFP FRET measurements. The described method can additionally be used to compare samples with very different fluorescence levels. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE).


Perez-Vilaro G.,University Pompeu Fabra | Sanjuan X.,University Pompeu Fabra | Sanjuan X.,Advanced Light Microscopy Unit | Diez J.,University Pompeu Fabra
Journal of Hepatology | Year: 2015

Background & Aims Decoding the myriad of interactions that hepatitis C virus (HCV) establishes with infected cells is mandatory to obtain a complete understanding of HCV biology and its associated pathogenesis. We and others have previously found that HCV infection disrupts the formation of P-bodies in cell culture. These are cytoplasmic RNA granules with key roles in post-transcriptional regulation of gene expression. Therefore, P-body disruption might have consequences beyond viral propagation. However, whether P-body disruption occurs also in vivo is unknown. Aim of this study was to address this important issue. Methods Formalin-fixed paraffin-embedded liver biopsies from four groups of patients (healthy donors, patients with non-virus related liver inflammation, HCV- and HBV-infected patients) were immunostained to detect DDX6 and Dcp1, two core P-body components. Changes in the localization of these proteins were assessed by confocal microscopy. Results HCV specifically inhibited P-body formation in hepatocytes from human livers regardless of viral genotype, inflammation grade or whether the infection was recent or long established. Importantly, this alteration was reversed once HCV was eliminated by therapy. Furthermore, we observed in vivo an unexpected heterogeneity in P-body composition, which might reflect functional specializations. Conclusions This is the first comprehensive in vivo P-body analysis that links a pathogenic condition to P-body alterations. Because of their role in gene expression, the alteration of P-bodies should be further studied to understand fully complex HCV-associated pathologies.


Pengo T.,University of Navarra | Pengo T.,Advanced Light Microscopy Unit | Munoz-Barrutia A.,University of Navarra | Ortiz-De-Solorzano C.,University of Navarra
IEEE Systems Journal | Year: 2014

Lung cancer is the deadliest form of cancer mainly because of the absence of reliable early diagnostic protocols. Therefore, there is increasing interest in the development of novel diagnostic noninvasive technologies that may improve the early detection of the disease. Bronchoscope-guided bronchoalveolar lavage (BAL) is a minimally invasive diagnostic technique that is based on the extraction and analysis of cellular material from the bronchial epithelium of patients that present suspicious lung masses on low-dose screening X-ray-computed tomography images. Together with a novel staining technique that combines immunophenotyping of a lung cancer biomarker with fluorescent in situ hybridization of genetically abnormal DNA loci, BAL promises a powerful early diagnostic tool for lung carcinomas. The sensitivity of this method, however, is highly dependent on the pathologist's ability to reliably and repeatedly examine thousands of cells under the microscope. This is an extremely labor-intensive and error-prone task. We have developed a multiscale multidimensional integrated microscopy computer-aided detection platform that autonomously scans and analyzes BAL samples. In this paper, we describe its software architecture and validate the specific image analysis protocols that are developed for this particular application. © 2014 IEEE.


Zimmermann T.,Advanced Light Microscopy Unit | Marrison J.,University of York | Hogg K.,University of York | O'Toole P.,University of York
Methods in Molecular Biology | Year: 2014

The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluorophores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given. © 2014 Springer Science+Business Media New York.


Agi E.,University of Texas Southwestern Medical Center | Langen M.,University of Texas Southwestern Medical Center | Altschuler S.J.,University of Texas Southwestern Medical Center | Wu L.F.,University of Texas Southwestern Medical Center | And 2 more authors.
Journal of Neurogenetics | Year: 2014

Visual systems have a rich history as model systems for the discovery and understanding of basic principles underlying neuronal connectivity. The compound eyes of insects consist of up to thousands of small unit eyes that are connected by photoreceptor axons to set up a visual map in the brain. The photoreceptor axon terminals thereby represent neighboring points seen in the environment in neighboring synaptic units in the brain. Neural superposition is a special case of such a wiring principle, where photoreceptors from different unit eyes that receive the same input converge upon the same synaptic units in the brain. This wiring principle is remarkable, because each photoreceptor in a single unit eye receives different input and each individual axon, among thousands others in the brain, must be sorted together with those few axons that have the same input. Key aspects of neural superposition have been described as early as 1907. Since then neuroscientists, evolutionary and developmental biologists have been fascinated by how such a complicated wiring principle could evolve, how it is genetically encoded, and how it is developmentally realized. In this review article, we will discuss current ideas about the evolutionary origin and developmental program of neural superposition. Our goal is to identify in what way the special case of neural superposition can help us answer more general questions about the evolution and development of genetically "hard-wired" synaptic connectivity in the brain. © 2014 Informa Healthcare USA, Inc.


PubMed | Advanced Light Microscopy Unit
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2013

The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluorophores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given.

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